System and apparatus for use in detecting microorganisms

ABSTRACT

A system and an apparatus for use in detecting a target microorganism or agent is disclosed which involves a solid support carrying a binding partner specific for the particular microorganism or agent and the solid support being characterised in that it defines means for protecting the binding partner from being dislodged or scraped off the solid support by physical means. The provision of protection against the binding partner being dislodged from or scraped off the solid support improves the reliability of tests such as immunoassays being conducted with the solid support and also enables such tests to be automated. Modules and machines for use with the solid support, and the automated conduct of tests ate also disclosed.

FIELD OF THE INVENTION

This invention relates to a system and an apparatus for use in detectinga target microorganism or agent. In one particular application of theinvention, the system and apparatus is used for the detection of lowlevels of a target microorganism (eg Salmonella) in the presence ofcompeting microorganisms.

BACKGROUND OF THE INVENTION

In the past few years, there has been a worldwide upsurge in the numberof reported outbreaks of food poisoning, often caused by Salmonellaalthough other bacteria such as Listeria have also been responsible forsome outbreaks. Listeria or Salmonella can be found as contaminants in awide variety of foods, particularly meat products; poultry; eggproducts; cheese, milk, icecream, and other dairy products; frozen andprocessed seafood; confectionary; and even vegetables and fruit.Listeria and Salmonella are recognised by food safety regulators in mostcountries of the world as being significant contaminants of food andmany government food safety regulators require environmental and endproduct testing for these bacteria, in the food industry. Consequently,it is common practice in the food industry to regularly check forcontamination by microorganisms of both food products and foodprocessing environments, such as Listeria and Salmonella. Similarly,testing for microorganisms is also carried out in other industries suchas pharmaceutical and cosmetics manufacturing.

Testing for microorganisms, generally involves taking a food sample (eg25 g portion) or a swab from the area being tested (nb samples may alsobe taken from floor sweepings, waste water and filtered air),transferring the sample to a pre-enrichment or enrichment medium inwhich any injured microorganisms will resuscitate, followed by one ortwo additional selective enrichment steps to increase the numbers of themicroorganisms of interest, and subsequent testing for the presence ofthe particular microorganisms in the medium using traditional culturalmethods or rapid methods such as immunoassays.

There are a number of known rapid methods for testing for Salmonella,Listeria and other pathogens, some of which are supplied by Tecra®International Pty Ltd of Frenchs Forest, New South Wales, Australia. Inone known Tecra® system, also described in Australian patentspecification No. 610925, a sample may be tested for, for example,Salmonella contamination by a method involving, firstly, transferringthe sample to a pre-enrichment medium for sixteen hours. A small aliquotof the pre-enrichment medium is then transferred to a first tube and adipstick which is coated with antibodies specific for Salmonella, isinserted into the first tube to capture any Salmonella microorganismspresent. After capture, which takes approximately twenty minutes, thedipstick is then washed in a second tube to remove any extraneousmaterial. The dipstick is then transferred to a third tube whichincludes a growth medium and any Salmonella which have attached to thedipstick multiply on the surface of the dipstick until they are presentin sufficient numbers for detection. For Salmonella, this replicationstage typically takes about four hours and after the four hourreplication period is over (different periods apply for differentmicroorganisms and different sample types), the dipstick is thentransferred to a fourth tube which contains enzyme-linked antibodiesspecific for Salmonella which bind to any Salmonella on the dipstick.The dipstick remains in the fourth tube for approximately thirtyminutes. The dipstick is then transferred to a fifth tube for washing toremove excess or unbound enzyme-linked antibodies. The dipstick is thentransferred to a sixth tube which contains substrate for the enzyme. IfSalmonella are present, a purple colour is produced on the lower half ofthe dipstick. A white band across the top of the dipstick acts as anegative control. The dipstick also incorporates a positive (purplecoloured) control as confirmation that the test has been carried outcorrectly.

Similar procedures to that described may be used for testing forListeria and for other selected microorganisms, although thepre-enrichment and growth media, incubation periods, incubationtemperature, number and timing of the various stages may vary frommicroorganism to microorganism.

Although the abovementioned test works well, the test involves numeroussteps that require a laboratory technician to monitor and time theprocedure and transfer the dipstick, to correct tubes, for the correctperiod, at the correct times, and at the correct incubation temperature,to ensure that the test is carried out properly.

The foregoing description of prior art, is not to be taken as anadmission that the art described forms part of the common generalknowledge of the person skilled in the art in Australia or elsewhere.

It is an object of the present invention to provide an improved systemand apparatus for detection of target microorganisms (eg bacteria suchas Salmonella and Listeria, and protozoa such as Cryptosporidium) and/oragents (eg viruses, prions, toxins, and other analytes includingantibodies, antigens, nucleic acids, chemical residues, microbialmetabolites and vitamins).

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a solidsupport for use in a process for the detection of a particular targetmicroorganism or agent and wherein the solid support carries a bindingpartner specific for the particular microorganism or agent, the bindingpartner being capable of selective capture and immobilisation of themicroorganism or agent characterised in that the solid support definesmeans for protecting the binding partner from being dislodged or scrapedoff the solid support by physical means.

In a preferred embodiment, the solid support is in the form of adipstick having a generally planar shape defining a longitudinal axis.

The dipstick may define a front face and a rear face. Typically themeans for protecting the binding partner from being dislodged from thedipstick includes at least one rail raised from the front face, andextending generally parallel to the longitudinal axis. More preferably,the means for protecting the binding partner from being dislodged fromthe dipstick includes a pair of such rails, in between which the frontface provides an array of regions, typically three or four, spaced apartalong the longitudinal axis. Typically, one of those regions willcomprise said binding partner, with two of the other regions providingpositive and negative controls.

The provision of protection against the binding partner being dislodgedfrom or scraped off the dipstick not only improves the reliability ofthe test, but also is a significant factor in allowing the process to beautomated. If the process of transferring the dipstick from tube to tubewhen carrying out the testing process is carried out by a machine, therisk of the dipstick brushing against the sides of one or more of thetest tubes is increased. If the rails were not present, such contactcould scrape the binding partner off the dipstick and potentiallycompromise the test.

In the preferred embodiment, the binding partners are simply applied tospecific regions on the dipstick which are preferably identified bynumbers or other suitable indicia.

However, in an alternative embodiment, the array of regions may bedefined by recesses in the front face of the dipstick. During themanufacture of the dipstick, the recesses may assist in locating andretaining droplets containing the binding partner (and substancesproviding the positive control) on the dipstick.

It is preferred that the rear face of the dipstick defines a pair ofribs which extend from the base of the dipstick towards the top of thedipstick and protrude from the rear face and increase in height relativeto the rear face as they extend towards the top of the dipstick.

The dipstick may define a lower portion for insertion into a well, tubeor the like, and an upper or handle portion to be grasped for moving thedipstick. Both upper and lower portions may define a through hole forchecking the location of the dipstick during the process of applying thebinding partner to the dipstick, and/or for locating the dipstick forreading results.

The dipstick may also define two flexible outwardly extending armsprojecting from opposite sides of the upper part of the lower portion ofthe dipstick.

Typically, the dipstick will be made out of a plastic which is resistantto gamma radiation to enable sterilisation of the surfaces of thedipstick in accordance with routine methods well known in the art.Preferably, the dipstick is made out of a polystyrene plastic. To assistin the reading of results, the dipstick is preferably of a substantiallyuniform white colour and has a substantially uniform level of opacity.

The binding partner (and/or substances providing positive and negativecontrols) may be adhered to the dipstick surface in a number of waysincluding hydrogen bonding and/or Van der Waals forces or by covalentbonds either directly or through a linker molecule. For example, thebinding partner may be conjugated to a biotin molecule and adhered tothe dipstick surface via an avidin or streptavidin linker molecule.

The binding partner may be any molecule or substance which specificallybinds to the target microorganism or agent. For example, for detectionof a target microorganism or a target protein or peptide, the bindingpartner is preferably selected from antibodies and antibody fragments(eg Fab and scFv fragments) which specifically bind to the targetmicroorganism or target protein or peptide. For a target protein orpeptide, the binding partner may also be a receptor molecule to whichthe target protein or peptide specifically binds. For detection ofantibodies, the binding partner may be an antigen or antigenicdeterminant for the target antibodies. For the detection of a nucleicacid (eg DNA or RNA), the binding partner may be selected from nucleicacids having a complementary nucleotide sequence such that the bindingpartner specifically hybridises to the target nucleic acid, preferablyunder conditions of high stringency. A nucleic acid binding molecule maybe adhered to the dipstick surface via, for example, a poly-dA probe.

In a particularly preferred embodiment, the dipstick is for use in aprocess for the detection of a particular target microorganism, and thebinding partner is an antibody specific for the particular microorganismwherein the binding partner is capable of selective capture andimmobilisation of the microorganism without compromising the ability ofthe microorganism to replicate.

A module is provided for use with the dipstick. The module defines astarting or “launch” slot for the dipstick, an end or “reading” slot anda series of wells or tubes therebetween. The shape and configuration ofthe module relative to the dipstick provides a number of key featuresand advantages.

It is preferred that the two opposed ends of the module have differentconfigurations. For carrying out a plurality of tests in parallel, atray may be provided on which a plurality of modules may be mounted andsecured in side by side relation. One end of the tray defines a firstseries of formations adapted to mate with only one of the ends of themodule, the other end of the tray defines a second series of formationsadapted to mate with the other of the ends of the module. This preventsany module being oriented “back to front” on the tray.

In a second aspect of the present invention, there is provided a modulefor use with the solid support of the present invention comprising astart slot, an end slot and a series of wells or tubes disposed betweenthe start slot and the end slot characterised in that at least the startslot defines a means to ensure that the solid support of the presentinvention can be inserted into the start slot in one orientation only.

Typically, the start slot, end slot and the wells are sized andconfigured, defining formations which interact with formations definedon the dipstick such that the dipstick may only be fully inserted in thestart slot, end slot and the wells in one orientation only. The meansfor ensuring that the solid support can only be inserted in oneorientation may include a pair of ribs which are spaced apart atapproximately the same distance as the rails of the dipstick. The ribsare preferably more closely spaced than the protruding ribs defined onthe rear face of the dipstick. Preferably, the width of the slots isgreater than the thickness of the dipstick but the width of the slotsplus the ribs defined in the slots is less than the thickness of thedipstick. Each of the wells defines a bulge or bulbous which is arrangedto face the reactive side of the dipstick in which the recesses arelocated but which is narrower than the dipstick.

It is preferred that the end slot of the module is configured such thatwhen the dipstick is inserted into that slot, the dipstick locks inplace and cannot be easily removed. This ensures that the dipstickscannot be deliberately or accidentally reused. The means may includecut-out portions in the slot into which the flexible outwardly extendingarms of the dipstick snap-fit.

The end slot of the module is also preferably provided with a windowthrough which the results obtained with the dipstick may be read eithermanually (ie by eye) or through automated means.

It is also preferred that the dipstick be provided with a frangibleportion to allow the upper portion of the dipstick to be “snapped” off.Removal of the upper portion of the dipstick when located in the endslot of the module allows for the wells to be readily sealed with, forexample, a strip of adhesive-backed foil or tape, for subsequentdisposal or for further assessment of the sample. That is, where thetest achieves a positive result for the presence of, for example,Salmonella, it may be desirable to subsequently confirm the result byplating out on agar an aliquot of the contents of a well within whichany Salmonella is grown (eg a “third” well including a growth medium).To assist with sealing of the wells of the module, the wells arepreferably provided with an upstanding lip upon which an adhesive-backedfoil or tape may be sealingly affixed.

In a related aspect, the present invention also provides a novel machinefor use with a dipstick and module of the present invention which ischaracterised by a reader means for reading the regions of the dipstick,said reader being arranged to move horizontally only in the machine,with the dipsticks being raised and lowered on a generally vertical axisto present the various regions of the dipstick to the reader means.

This arrangement makes the machine simpler to construct control andoperate as the reader means, typically comprising a light or reflectancedetector (eg a CCD or photopic sensor) and one or more light sources (egLED(s)), only has to move in a horizontal direction.

The present invention also provides a machine which may be used with adipstick and module of the present invention which is characterised by areader means for reading the regions of the dipstick, said readercomprising a light or reflectance detector and one or more lightsources.

The light source(s) used in the reader means preferably comprises a pairof LED's arranged so as to uniformly illuminate the dipstick in theregion of the front face from where the results are to be read. Each LEDmay provide a light band within the range of about 20 to 40°, morepreferably about 30° and may be placed at an angle to the front face ofthe dipstick which is in the range of about 60 to 80′, more preferablyabout 70′.

The present invention further provides a novel machine for use with adipstick and module of the present invention which is characterised bythe dipstick being automatically and sequentially moved to and loweredinto and raised from wells or tubes in the module in sequence with thedipstick remaining in each well for a predetermined period of time.

Automatic movement of the dipstick rather than say the liquidsassociated with the assay makes operation of the system easier and morereliable.

The machine may include a head defining a gripper means for grasping atop portion of the dipstick. The head is preferably adapted tosimultaneously grasp the top portion of more than one dipstick, suchthat the machine may simultaneously move dipsticks between the slots andwells or tubes of respective modules so as to allow simultaneous andmultiple assays to be conducted.

It is preferred that the movement of the head and hence the dipstick maycontrolled to suit particular assays being carried out by the machine.This is preferably achieved through the use of a smartcard and smartcardreader.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

A specific embodiment of the invention as applied to the detection of atarget microorganism will now be described, with reference to theaccompanying figures in which:—

FIG. 1 is a perspective view of a dipstick embodying the presentinvention;

FIG. 2 is a front view of the dipstick of FIG. 1;

FIG. 3 is side view of the dipstick of FIG. 1;

FIG. 4 is a rear view of the dipstick shown in FIG. 1;

FIG. 5 a is a top end view of the module associated with the dipstick;

FIG. 5 b is a bottom end view of the module associated with thedipstick;

FIG. 6 a is a side elevation of the module associated with the dipstick;

FIG. 6 b is an opposite side elevation of the module associated with thedipstick;

FIG. 7 a is an end elevation of one end of the module of FIGS. 5 a andb;

FIG. 7 b is an opposite end elevation of one end of the module of FIGS.5 a and b;

FIG. 8 a is a perspective view of the module;

FIG. 8 b is a perspective view of the dipstick inserted in a slot of themodule;

FIG. 9 is a perspective view of a first part of a gripper which engagesthe dipstick in the automated immunoassay machine;

FIG. 10 is a perspective view of the assembled gripper comprising firstand second parts;

FIG. 11 is an exploded perspective view of a “multi-gripper” comprisingtwo metal, preferably aluminium, bars, machined to provide slots for upto 30 dipsticks, the two bars incorporating 30 springs, one for eachgripper position. The two bars are screwed together to form a complete“multi-gripper” assembly.

FIG. 12 a illustrates an automated immunoassay machine;

FIG. 12 b illustrates an automated immunoassay machine with the frontdoor in the open position; and

FIG. 13 schematically illustrates an optical reader of an automatedimmunoassay reader, and the arrangement thereof relative to a dipstick.

DETAILED DESCRIPTION OF THE INVENTION

Referring to the drawings, FIG. 1 shows a solid support in the form of adipstick 10. The dipstick is generally elongate and planar. The dipstickis preferably made from a “general purpose” polystyrene plastic and isof a substantially uniform white colour and has a substantially uniformlevel of opacity. The dipstick is for insertion into wells of a module100 illustrated in FIGS. 5 to 8 described in more detail below. Thedipstick defines a lower part 14 which in use is lowered into wells orslots defined in the module and an upper part 12 which in use, isgrasped by an automated immunoassay machine 300 (shown in FIGS. 12 a andb) which is programmed to move the dipstick into the various wells ofthe module according to a programmed sequence of operations.

The lower part of the dipstick 14 has a front face 14 a best seen inFIGS. 1 and 2 and a rear face 14 b best seen in FIG. 4. The lowerportion 14 of the dipstick is a generally rounded base 16 and sides 18and 20 which gradually taper outwardly from the base. Two parallelelongate rails 22 extend from dose to the base 16 of the lower portionto a frangible portion 15 separating the lower portion 14 from the upperportion 12. As is best seen in FIG. 1, a series of four regions 24 aredefined in the front face of the dipstick between the rails 22. Theregions which in the preferred embodiment are indicated by the numbers“1”, “2”, “3”, and “4” comprise part of the chemistry of a process fordetecting microorganisms. In particular, the two uppermost regions “1”and “2” comprise the chemistry necessary for positive and negativecontrols. Two of the other regions “3” and “4” comprise, for example,highly specific purified antibodies (such as, monodonal antibodies) toselectively capture a target microorganism, such as Salmonella orListeria. One or both of the regions may be used. There is a relativelylarger space between regions “3” and “4”.

This specification is not specifically concerned with the chemistry ofthe process, rather with apparatus for carrying out the process. Theskilled person can turn to Australian patent specification No. 610925,the contents of which are incorporated herein by reference, for adetailed discussion of the chemistry of the process.

The reverse side 14 b of the dipstick, (best seen in FIG. 4) is alsogenerally planar and also defines spaced apart ribs 26. In contrast withthe ribs on the front of the dipstick, ribs 26 taper inwardly from thebase 16 of the dipstick towards a frangible portion 15. The ribs 26 arealso spaced apart at a greater distance relative to the ribs 22. Asshown in FIG. 3, the height or thickness of the ribs gradually increasesfrom the base 16 as the ribs extend towards the upper part of thedipstick. A through hole 28 extends through the dipstick near thefrangible portion 15.

A pair of outwardly curved flexible arms 30 extend away from either sideof the dipstick, a gap 32 being defined between each arm of the dipstickand the dipstick itself. The arms are relatively flexible and may bendtowards the central portion of the dipstick to dose or partly dose thegap 32.

The upper part of the dipstick 12 is configured to engage with a gripperof the machine. Two embodiments of the gripper are shown in the figures.First, a single gripper is shown in FIGS. 9 and 10, and second, a“multi-gripper” is shown in FIG. 11. As seen in FIG. 1, a flexibletongue portion 34 is defined in an elongate aperture in the upperportion 36. A hemispherical protrusion 38 is defined on a free end ofthe tongue 36, best seen in FIG. 4. The front face of the upper portionof the dipstick defines an outwardly expanding flared recess 39. Therecess 39 and tongue 34 are used to align and secure the dipstick in thegripper.

A hole 42 is defined in the upper portion to one side of the centralaxis A of the dipstick. This is used to locate the dipstick during theapplication of the binding partner (and substances providing positiveand negative controls) during manufacture of the dipstick. It is alsoused to provide a reference point for the reader of the machine of theinvention, to assist in reading results from the dipstick.

FIGS. 5 to 8 illustrate the module 100 which is used with the dipstick.The module comprises an initial dipstick location slot 102 at one end ofthe module, an end slot 116 and a series of six wells 104, 106, 108,110, 112, 114 “tubes 1 to 6” disposed in a line between the start slotand the end slot. Each of the wells are provided with an upstanding hp124 (best seen in FIG. 8 a) to allow easy sealing with anadhesive-backed foil or tape. The device is moulded in a plastic that isresistant to gamma radiation. The module may be provided with a pair offeet 126 which may reduce flex in the module (which may assist in thesealing operation(s) of the module) and be shaped to provide a “grip”for automated manufacturing processes. The shape and configuration ofthe module and the start and end slots and the six wells is such as toenable satisfactory automation of the process, as follows.

First, the shape of the front 118 of the module and the rear 120 of themodule is different. When tests are being carried out by automation, themodules are located in side by side relation in stainless steel trays,so that the automated immunoassay machine 300 (shown in FIGS. 12 a andb) can carry out a number of assays in parallel at the same time. Thedifferent configuration of the front and rear portions of the module inconjunction with the configuration of the tray, ensures that the trayscan only be loaded in the tray in one direction. This is important asthe process only works if the steps are carried out in the correctorder.

At the end of the process, the results are read in the machine by anoptical reader. Accordingly, it is important that the dipstick becorrectly oriented so that it faces the correct direction at the end ofthe testing procedure with the regions facing the optical reader. Asseen in FIGS. 5 a and b, two spaced apart rails project into theinterior of the initial slot 102 from the back wall of the slot Theserails are spaced apart at the same distance as the rails 22 projectingfrom the front of the dipstick. Identical pairs of rails project intothe interior of each of the wells and the end slot from their respectivefront walls. These rails widen tapering outwardly as they extend downinto the slots/wells. This is best seen in FIGS. 7 a and b, which showsthe rails in the end slot 120. The width of the slot 102, measured fromthe front wall 102 a to the rear wall 102 b in the direction of thelongitudinal central axis of the module, is greater than the thicknessof the dipstick 10 taking into account both the front ribs 22 and thetapered ribs 26. However, the width of the slot minus the width of theribs 122 is narrower than the thickness of the thicker parts of thedipstick where the tapering ribs 26 are relatively thicker. Thus,because the rails 22 are spaced apart the same distance as the ribs 122,it is impossible to properly insert the dipstick in the module with ribs22 facing the ribs 122 of the module as the effective width of the slotis less than the thickness of the dipstick. The further the dipstick isinserted into the front slot in this orientation, the more difficultinsertion becomes, the ribs 26 gradually increase in thickness. However,if the dipstick is inserted with the outwardly tapering ribs 26 facingthe front of the module, those ribs, being spaced wider apart than ribs122, locate either side of those ribs, and the dipstick can be fullyinserted into the starter slot 102. Insertion is assisted since the ribs26 taper outwardly at approximately the same angle as the ribs 122.

Each of the subsequent six wells of the module comprises a slot portionwhich is essentially identical to the initial slot portion, including,as discussed, the provision of the outwardly tapering ribs but includesan outwardly extending bulbous portion 130 which allows an increasedvolume of fluid (reagent) to be located in the well as compared with thefront slot 102, whilst still providing the same protection against thedipstick being incorrectly inserted since the side portions of the wellsare essentially the same width as the starter slot. The bulbous portionalso allows more reagent to come into contact with the front face of thedipstick particularly when the dipstick is jiggled in the well. This isbest seen in FIGS. 6 a and b. The uppermost parts of the wells taperoutwardly and this is to receive the outwardly extending arms 32 of thedipstick.

The end slot also defines two outwardly tapering ribs but also as shownin FIG. 7 b, the rear wall of the end slot is substantially cut away toallow the dipstick to be viewed from the rear of the module when thedipstick is located in the end slot. This allows an optical readercomprising a CCD, photopic sensor or the like to be used to read thedipstick to assess the colour of the dipstick to see whether there is apositive or negative result from the test. This process, which inAustralian patent specification No. 610925 is done by eye, may beautomated in the machine 300.

To prevent reuse of the dipstick, and to lock the dipsticks into themodule to allow separation of the machine from the dipsticks as seen inFIG. 6 a, two cut out portions 140 are defined in the upper parts of theside walls of the end slot. When the dipstick is fully inserted into theend slot, the flexible arms 32 are initially compressed inwardly andthen when the top 32A of each arm drops below the rim of the module, thearms expand outwardly into the cut-out portions 140. Any attempt toremove the dipstick by pulling it upwardly, damages the dipstick bycausing the arms to break.

The dipstick includes a frangible portion 15 to allow the upper andlower portions of the dipstick to be separated by bending or snapping.Removal of the upper portion of the dipstick when located in the endslot of the module allows for the wells to be readily sealed with anadhesive-backed foil or tape.

The gripper assembly 200 shown in FIGS. 9 and 10 comprises two discreteparts, namely a first part 201 and a second part 202. The two parts arejoined by a screw (not shown) passing through hole 204, and are alignedby studs (not shown) and receiving apertures 203. A slot 205 is definedbetween the two parts of the gripper into which the upper portion 12 ofthe dipstick locates The slot is configured to mate with the top of thestick.

In particular, the flexible tongue 34 of the dipstick is received withinan elongate channel 206 defined on part 201 and the flared recess 39locates around a generally trumpet shaped formation 208 on part 201formed between the first and second parts.

When the tongue 34 is located in the slot, the hemispherical protrusion38 locates in a recess (not shown) in part 202 and acts to retain thetongue 34 within the slot and, as a consequence, the upper portion 12 ofthe dipstick remains gripped by the gripper assembly 200.

The “multi-gripper” assembly shown in FIG. 11 comprises two metal,preferably aluminium, bars 400 a and 400 b, machined to provide slotsfor up to 30 dipsticks, the two bars incorporating 30 springs (eg 401),one for each gripper position. The two bars are screwed together to forma complete “multi-gripper” assembly. The multi-gripper enables multipledipsticks, normally up to 30, to be gripped at the same time, and movedbetween the slots, wells or tubes of respective modules.

An optical reader 500 is shown in FIG. 13. The optical reader comprisesa pair of LED's (501 and 502) angled at about 72′ to the front face 503of the dipstick 504. For assays involving the use of alkalinephosphatase and BCIP/NBT (Bromochloroindolyl phosphate/nitrobluetetrazolium) substrate, the purple coloured positive results are bestread using LED's emitting green light, preferably of about 510 to 590nm, more preferably of about 530 to 570 nm, most preferably about 530nm. Suitable LED's are available from Agilent (USA), Kingbright (Taiwan)and Toyoda (Japan). The pair of LED's (501 and 502) preferably providelight of an intensity of about 8 to 12 candellas incident on the frontface 503 of the dipstick 504. The light band emitted by the LED's may bewithin the range of about 20 to 40°, more preferably about 30°. TheLED's are arranged on either side of a reflectance collecting tube 505for the photopic sensor 506. The collecting tube 505 is about 30 mm inlength and has an outer diameter of about 1.6 mm and an internaldiameter of about 1.2 mm. The collecting tube 505 is preferablyinternally etched with phosphoric acid and painted or coated with a mattblack finish. The front of the collecting tube 507 is best locatedbetween about 50 and 70 mm from the front face 503 of the dipstick 504.

During the process of manufacturing of dipsticks for detection of atarget microorganism, antibodies specific to the microorganism areapplied to one or two of the regions 24, typically region “4” or, ifneeded, region “3”. One of the other regions comprise substances forpositive control, typically region “1”, and region “2” typicallycomprises a negative control.

In use, the process for carrying out automated tests for Salmonella areas follows.

The modules and dipsticks are either warmed or allowed to reach roomtemperature. A stainless steel tray is removed from the automatedimmunoassay machine 300 placed on the bench and modules are locked intothe tray. The tray defines numbered positions and the modules are placedon the tray according to the numbers on the tray as instructed by themachine. The modules are supplied containing all the necessary reagents,and washes with the wells sealed with foil to retain and protect thereagents apart from the sample itself, which is loaded into buffer inthe first well of the module, after the foil covering the wells has beenremoved from the module. The dipstick is then inserted into the firstslot at the front of each module in the correct orientation assisted bythe provision of the various ribs. The tray is then placed into theautomated immunoassay machine. Once the test has begun, the“multi-gripper” comprising an aluminium arm inside the machinecomprising two bars defining a series of gripper slots, is moveddownwards onto the dipsticks and the grippers grab the upper portions 12of the dipsticks. The dipsticks are lifted by the arm from the startingslots and may be initially moved to the back of the machine, that is, tothe end slots, where they can be read by an optical reader to establisha background signal for each dipstick.

The dipsticks are moved to “tube one”, which contains the sample to betested, for a predetermined period of time, which for the Salmonellatest is, typically, twenty minutes. At this stage, the dipsticks may beraised and lowered inside the tubes to “jiggle” them and ensure that thecontents of the tube are mixed. In the case of a Salmonella test, thisalso ensures that the buffer additive, initially located in tube one, ismixed into the sample which is added to tube one.

The arm is then raised and the dipsticks are moved out of “tube one” andlowered into “tube two” for washing in a wash solution (eg modifiedbuffered peptone water; MBPW) and are washed by moving the dipsticks upand down.

The arm is then raised and the dipsticks are lifted out of tube two andloaded into tube three which contains a growth medium (eg in the case ofSalmonella, this may be M broth). The dipsticks remain in tube typicallyfor three to four hours during which the temperature within the tube twois raised to an appropriate culture temperature by a heater unit (eg aflexible heating mat) within the automated immunoassay machine 300.Occasional jiggling of the dipsticks may be carried out.

The arm is then raised again and the dipsticks are lifted out of tubethree and lowered into tube four which contains an enzyme-linkedantibody conjugate. Typically, the dipsticks remain here for thirtyminutes. The arm is then raised and the dipsticks are lifted out of tubefour and lowered into tube five which contains a wash solution. Thedipsticks are washed for ten minutes with the arms continuously raisingand lowering the dipsticks to ensure that the dipsticks are properlywashed.

The arm is then raised, lifting the dipsticks out of tube five, and thedipsticks are then lowered into tube six which contains about 1 ml ofsubstrate for the enzyme. For Salmonella, the dipstick will remain inthis tube for about ten minutes.

The arm is then raised and the dipsticks are lifted out of tube six,re-aligned by moving back to the starting slot 102 and then insertedinto the end slot 116 (ie the “reading slot”) of the module. The opticalreader then moves into line with the dipstick to read the dipstick inthe first module. The dipstick is moved so that the reader detects thehome position defined by the hole 28. The dipstick is then raised sothat the optical reader can read the negative control area, the positivecontrol area and the test area. The optical reader then moves until itis level with the dipstick associated with the second module andperforms the same process and so on with the third module until all thedipsticks are read. It is important to note that because the dipstickscan move up and down in a vertical direction, the optical reader onlyneeds to be able to move along a horizontal axis. The arm is thenlowered so that the dipsticks are pushed as far down into the tray asthey can go which drops the curved arms 32 into the modules (and alsoinhibits reuse of the dipsticks).

The arm is then raised and the dipsticks which are now locked into themodules separate from the multi-gripper.

Difficulties in the optical reader correctly reading results may beexperienced if the dipstick is not placed in the end slots such that thefront face 14 a of the dipstick is not substantially vertical (ie suchthat region “4” of the dipstick may be relatively doser or further awayfrom the optical reader than region “1”). To overcome such a difficulty,the background reading scan may be used to establish a baseline betweenreadings either side of each of regions “1”, “2”, “3” and “4”, and theresult readings may be subjected to an algorithm that calculates:

-   -   (i) For the positive control (PC) region (typically region “1”),        the maximum or total decrease in reflectance from the PC        baseline;    -   (ii) For the negative control (NC) region (typically region        “2”), the maximum increase in reflectance from the NC baseline;        and    -   (ii) For the sample (S) region (typically region “4”), the        maximum or total decrease in reflectance from the S baseline.

For other microorganisms such as Listeria, the tests are run slightlydifferently with different timing and different reagents. Accordingly,the machine may have a smartcard reader 302, for receiving smartcardsparticular to certain microorganisms or for specific assay protocols,for example testing for Salmonella in confectionary involves a differentprotocol to testing for Salmonella in a different type of food product.The smartcards program the machine to perform the test in the mannerappropriate for the particular microorganism.

The system is not limited to testing for bacteria such as Listeria andSalmonella. Any microorganism which is capable of in vitro growthincluding yeasts, moulds, protozoa (eg Cryptosporidium) and otherbacteria (eg Escherichia coli, Legionella, Campylobacter,Staphylococcus, Bacillus and Pseudomonas), can be tested for. Thebinding partner for capture of such microorganisms is preferablyselected from antibodies or antibody fragments (eg Fab and scFvfragments) which specifically bind to an antigenic determinant or haptenon the surface of the particular target microorganism, for example anantigenic determinant present in a cell wall protein such as a porin orin a flagellal protein. For detection of a captured targetmicroorganism, typically an enzyme-linked antibody conjugate is usedwhich specifically binds to any antigenic determinant or hapten presentin a cell wall protein such as those mentioned above or, otherwise, in asecreted protein such as a toxin (eg enterotoxins of Bacillus andStaphylococcus, and emetic toxins of Bacillus).

The system can also be used to capture and detect other agents that maynot require a growth step to enable detection. Such other agents includeviruses (eg HIV, HCV, etc), prions (eg BSE), toxins (eg enterotoxins ofBacillus and Staphylococcus, and emetic toxins of Bacillus), and otheranalytes including antibodies, antigens (eg food allergens such as milkproteins including caseins, and peanut proteins), nucleic acids,chemical residues (eg antibiotic and pesticide residues), microbialmetabolites (eg mycotoxins and phycotoxins), and vitamins.

The system can also be operated manually. In such cases, the modules maybe held in a plastic tray, with the dipsticks being moved between theslots and wells of the module by hand, and the modules being moved inand out of an incubator as required. The results may be read by eye andcomparison against a standard colour chart.

EXAMPLE 1 Analysis of Salmonella Using a Salmonella Assay Dipstick andModule

Materials and Methods

Gamma-irradiated white polystyrene plastic dipsticks as shown in FIG. 1,were prepared by coating an antigen solution (ie 10 μl protein extractsof Salmonella bacteria) onto region 1 (ie to provide a positivecontrol), and a capture antibody solution (ie 10 μl affinity-purifiedantibody recognising Salmonella) onto regions 3 and 4 (ie for samplebinding). These solutions were prepared in buffer using standardprocedures. The solutions were air-dried, and then the dipsticks wereincubated in a solution of a protein blocking agent (eg casein oralbumin) in buffer. The dipsticks were then air-dried again and sealedwith a dessicant sachet in a foil pouch for storage at 4C prior to use.

Assay modules having a configuration as shown in FIGS. 5-7, and whichwere suitable for enriching for and assaying for Salmonella wereprepared substantially in the manner described in Australian patentspecification No. 610925. Tubes 1 to 6 were filled as follows:

-   -   Tube 1: 0.75 ml sample additive (as per Tecra® Salmonella        Unique™ assay protocol)    -   Tube 2: peptone-buffered water wash solution (1.5 ml) (MBPW)    -   Tube 3: enrichment broth for the enrichment of Salmonella (1 ml)    -   Tube 4: enzyme-linked antibody conjugate (ie which recognises        Salmonella bacteria) in buffer solution (1 ml)    -   Tube 5: wash solution (1.5 ml)    -   Tube 6: substrate solution (ie BCIP/NBT) for the enzyme of the        enzyme-linked antibody conjugate of tube 4 (1 ml)

The tubes were sealed with an aluminium foil seal.

For analysis of Salmonella in various food matrices, 10 g samples weretaken and mixed with 90 ml sterile modified peptone buffered water(MBPW), and pre-warmed to room temperature. Each sample was then mixedand sealed for incubation at 35-37° C. for 16-20 hrs (Pre-enrichmentstep). Additional samples were prepared by spiking Salmonella speciesdirectly into the food. Spikes were prepared at a level of 10 cells perfood sample. The spiked food samples were chilled before extraction withMBPW.

The aluminium foil seal on each assay module was removed, and a 1 mlsample from a pre-enrichment sample was each transferred to the tubes 1and a dipstick inserted into each of the launch slots. Each module wasplaced in a tray which was then inserted into the immunoassay machineshown in FIG. 12 and the machine programmed with a Salmonella assaysmartcard. The programmed assay involved:

(i) Moving the dipsticks to tubes 1 for 20 minutes (42° C.) to captureany Salmonella present with the capturing antibody.

(ii) Moving the dipsticks to tubes 2 for 7 minutes (42° C.), withjiggling, for a first wash to remove unbound material.

(iii) Moving the dipsticks to tubes 3 for 4 hours (42° C.), to allow thecaptured bacteria to grow (enrichment step).

(iv) Moving the dipsticks to tubes 4 for 30 minutes (room temperature)to allow specific binding of the detection antibody (ie theenzyme-linked antibody conjugate).

(v) Moving the dipsticks to tubes 5 for 10 minutes (room temperature),with jiggling to wash to remove unbound material.

(vi) Moving the dipsticks to tubes 6 for 10 minutes (room temperature)to allow development of purple coloured results.

(vii) Moving the dipsticks to the end slots for reading of the results.

The machine was activated and run for a total assay time of 5 hr 45 min.At the end of this period, the sample results were printed or downloadedto a computer. The tray containing the modules was then removed and thedipsticks examined visually to confirm the results. Samples taken fromthe tubes 3 were streaked onto XLD and HE plates (Oxoid Unipath, UnitedKingdom) to confirm the presence of Salmonella in positive samples,using conventional procedures.

Results

The results are shown in Table 1. TABLE 1 Confirmation of result UniquePlus Instrument Enrichment Tube 3 Sample and Result Stick confirmationconfirmation Sample Bacteria Spike Result Visual XLD HE XLD HE Sultanasnil 0 −ve −ve No No No No growth growth growth growth S. dublin 10 + +Typical Typical Typical Typical S. dublin 10 + + Typical Typical TypicalTypical Brazil Nuts nil 0 −ve −ve No No No No growth growth growthgrowth S. bredeney 10 + + Typical Typical Typical Typical S. bredeney10 + + Typical Typical Typical Typical Roasted nil 0 −ve −ve No No No NoPeanuts growth growth growth growth S. bredeney 10 + + Typical TypicalTypical Typical S. bredeney 10 + + Typical Typical Typical TypicalCherries nil 0 −ve −ve No No No No growth growth growth growth S. 10 + +Typical Typical Typical Typical montevideo S. 10 + + Typical TypicalTypical Typical montevideo Raspberries nil 0 −ve −ve No No No No growthgrowth growth growth S. dublin 10 + + Typical Typical Typical Typical S.dublin 10 + + Typical Typical Typical Typical

EXAMPLE 2 Analysis of Listeria

Materials and Methods

Gamma-irradiated white polystyrene plastic dipsticks as shown in FIG. 1,were prepared by coating an antigen solution (ie 10 μl protein extractsof Listeria bacteria) onto region 1 (ie to provide a positive control),and a capture antibody solution (ie 10 μl affinity-purified antibodyrecognising Listeria) onto regions 3 and 4 (ie for sample binding).These solutions were prepared in buffer using standard procedures. Thesolutions were air-dried, and then the dipsticks were incubated in asolution of a protein blocking agent in buffer. The dipsticks were thenair-dried again and sealed along with a dessicant sachet in a foil pouchfor storage at 4C prior to use.

Assay modules having a configuration as shown in FIGS. 5-7, and whichwere suitable for enriching for and assaying for Listeria were preparedsubstantially in the manner described in Australian patent specificationNo. 610925. Tubes 1 to 6 were filled as follows:

-   -   Tube 1: no solution.    -   Tube 2: no solution (this tube is not used in the Listeria        assay)    -   Tube 3: enrichment broth for the growth of Listeria (1 ml)    -   Tube 4: enzyme-linked antibody conjugate (ie which recognises        Listeria bacteria), in buffer solution (1 ml)    -   Tube 5: wash solution (1.5 ml)    -   Tube 6: substrate solution (BCIP/NBT) (1 ml)

The tubes were sealed with an aluminium foil seal.

Analysis of Listeria was conducted in a similar manner to that employedfor Salmonella in Example 1.

Cultures of Listeria were prepared containing 10⁶ cells/ml and a 1 mlsample of each culture introduced to tube 1 of a module from which thefoil seal had been removed. Dipsticks were then inserted into the launchpositions of each module, and each module inserted into an assay tray.Additional samples were prepared and their modules inserted into thetray alongside. The tray containing the modules was inserted into theimmunoassay machine which had been programmed with a smartcard foroperation of a Listeria assay protocol. The protocol did not involvedipping the dipsticks into the tubes 2. The programmed assay involved:

(i) Moving the dipsticks to tubes 1 for 1 hour (31° C.) to capture anyListeria present with the capturing antibody.

(ii) Moving the dipsticks to tubes 3 for 5 minutes (31° C.), to allowthe captured bacteria to grow.

(iv) Moving the dipsticks to tubes 4 for 30 minutes (42° C.) to allowspecific binding of the detection antibody (ie the enzyme-linkedantibody conjugate).

(v) Moving the dipsticks to tubes 5 for 10 minutes (room temperature),with jiggling to wash to remove unbound material.

(vi) Moving the dipsticks to tubes 6 for 15 minutes (room temperature)to allow development of purple coloured results.

(vii) Moving the dipsticks to the end slots for reading of the results.

The machinne indicated a total assay time of 7 hours, and at the end ofthis period, the sample results were printed. The tray containing themodules was then removed and the dipsticks examined visually to confirmthe results.

Results

The results are show in Table 2. TABLE 2 Dipstick Name Result 1 L.monocytogenes Positive 2 L. monocytogenes Positive 3 L. innocua 6APositive 4 L. innocua 6B Positive 5 L. innocua Positive 6 L. innocuaPositive 7 L. innocua Positive 8 L. innocua Positive 9 L. innocuaPositive 10 L. innocua NC Error 11 L. innocua Positive 12 L. innocnaPositive 13 L. innocua Positive 14 L. seeligeri Positive 15 L. seeligeriNegative 16 L. seeligeri Negative 17 L. seeligeri NC Error 18 L.seeligeri Positive 19 L. welshimeri 4 Positive 20 L. welshimeri 4Positive*NC error is reported by the assay when the dipstick is unreadable forany reason. (Dipstick 10, this was repeated at a later date).

Example 3 Analysis of Staphylococcal Enterotoxins

Materials and Methods

Gamma-irradiated white polystyrene plastic dipsticks as shown in FIG. 1,were prepared by coating an antigen solution (ie 10 μl enterotoxinprotein) onto region 1 (ie to provide a positive control), and anantibody solution (ie 10 μl of affinity-purified capture antibodyrecognizing S. aureus enterotoxin) onto regions 3 and 4 (ie for samplebinding). These solutions were prepared in buffer using standardprocedures. The solutions were air-dried, and then the dipsticks wereincubated in a solution of a protein blocking agent in buffer. Thedipsticks were then air-dried again and then sealed along with adessicant sachet in a foil pouch for storage at 4° C. prior to use.

Assay modules suitable for S. aureus enterotoxin detection wereprepared. Tubes 1 to 6 were filled as follows:

-   -   Tube 1: no solution    -   Tube 2: wash solution (1.5 ml)    -   Tube 3: wash solution (1.5 ml)    -   Tube 4: enzyme-linked antibody conjugate (ie which recognises S.        aureus enterotoxin) in buffer solution (1 ml)    -   Tube 5: wash solution (1.5 ml)    -   Tube 6: substrate solution (BCIP/NBT) (1 ml)

The tubes were sealed with an aluminium foil seal.

Samples were extracted from food by mixing 10 g of food with 25-50 mlbuffer using the methodology described in the Tecra® Visual Immunoassay(VIA™) kit instruction book (Tecra International Pty Ltd, FrenchsForest, New South Wales, Australia).

An aliquot of each sample (1 ml) is introduced into tube 1 of eachmodule together with a sample additive (as detailed in the VIA™ kitinstructions) and the dipsticks inserted into the launch positions. Themodules were then placed into an assay tray for loading into animmunoassay machine. The immunoassay machine was programmed with asmartcard for operation of an enterotoxin analysis assay. The programmedassay involved:

-   -   (i) Moving the dipsticks to tubes 1 for 2 hours (35-37° C.) to        capture any enterotoxin present with the capturing antibody.

(ii) Moving with the dipsticks to tubes 2 for 2 minutes (28-30° C.),with jiggling, for a first wash to remove unbound material.

(iii) Moving the dipsticks to tubes 3 for 2 minutes (28-30° C.), withjiggling, for a further wash.

(iv) Moving the dipsticks to tubes 4 for 1 hour (28-30° C.) to allowspecific binding of the detection antibody.

(v) Moving the dipsticks to tubes 5 for 5 minutes (2830° C.) to wash toremove unbound material.

(vi) Moving the dipsticks to tubes 6 for 10 minutes (room temperature)to allow development of purple coloured results.

(vii) Moving the dipsticks to the end slots for reading of the results.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

1. A solid support for use in a process for the detection of aparticular target microorganism or agent and wherein the solid supportcarries a binding partner specific for the particular microorganism oragent, the binding partner being capable of selective capture andimmobilisation of the microorganism or agent characterised in that thesolid support defines means for protecting the binding partner frombeing dislodged or scraped off the solid support by physical means. 2.The solid support of claim 1, wherein the solid support is in the formof a dipstick having a generally planar shape defining a longitudinalaxis.
 3. The solid support of claim 2, wherein the solid support definesa front face and a rear face and the means for protecting the bindingpartner from being dislodged from the solid support includes at leastone rail raised from the front face and extending generally parallel tothe longitudinal axis, and wherein adjacent to said rail, the front faceprovides an array of regions spaced apart along the longitudinal axis.4. The solid support of claim 2, wherein the solid support defines afront face and a rear face and the means for protecting the bindingpartner from being dislodged from the solid support includes a pair ofrails raised from the front face and extending generally parallel to thelongitudinal axis, and wherein between each rail of the said pair ofrails, the front face provides an array of regions spaced apart alongthe longitudinal axis.
 5. The solid support of claim 3, wherein saidarray of regions comprises 3 or 4 regions, at least one of which hassaid binding partner applied thereto.
 6. The solid support of claim 3,wherein the rear face of the solid support defines a pair of ribs whichextend in a direction generally parallel to the longitudinal axis fromthe base of the solid support to the top of the solid support andprotrude from the rear face.
 7. The solid support of claim 6, whereineach of the ribs of said pair of ribs increases in height relative tothe rear face as they extend towards the top of the solid support. 8.The solid support of claim 2, wherein the solid support defines a lowerportion for insertion into a well, tube or the like, and an upper orhandle portion to be grasped for moving the solid support.
 9. The solidsupport of claim 8, wherein the solid support defines two flexibleoutwardly extending arms projecting from opposite sides of an upper partof the lower portion of the solid support.
 10. The solid support ofclaim 8, wherein the solid support is provided with a frangible portionto allow the upper portion of the solid support to be snapped off. 11.The solid support of claim 1, wherein the solid support is of asubstantially uniform white colour and has a substantially uniform levelof opacity.
 12. The solid support of claim 1, wherein the bindingpartner is selected from the group consisting of antibodies and antibodyfragments, receptor molecules, antigens or antigenic determinants, andnucleic acids.
 13. The solid support of claim 1, wherein the solidsupport is intended for use in a process for the detection of a targetmicroorganism, and the binding partner is an antibody specific for theparticular microorganism, and wherein the binding partner is capable ofselective capture and immobilisation of the microorganism withoutcompromising the ability of the microorganism to replicate.
 14. A modulefor use with the solid support of claim 1, the module comprising a startslot, an end slot and a series of wells disposed between the start slotand the end slot, said start slot, end slot and wells being adapted toreceive said solid support, and wherein the module is characterised inthat at least the start slot defines a means to ensure that the solidsupport can only be inserted into the start slot in one orientation. 15.The module of claim 14, wherein the start slot end slot and the wellsare sized and configured defining formations which interact withformations defined on the solid support such that the solid support mayonly be inserted in the start slot, the end slot and the wells in oneorientation.
 16. A module for use with the solid support of claim 4, themodule comprising a start slot, an end slot and a series of wellsdisposed between the start slot and the end slot, said modulecharaterised in that the start slot, end slot and the wells are sizedand configured defining formations which interact with formationsdefined on the solid support such that the solid support may only befully inserted in the start slot, the end slot and the wells in oneorientation, and wherein said formations defined by the start slot, endslot and the wells include a pair of ribs which are spaced apart atapproximately the same distance as the rails provided on the solidsupport.
 17. The module of claim 14, wherein the end slot is configuredsuch that upon insertion of the solid support into that slot, the solidsupport locks in place and cannot be easily removed.
 18. A module foruse with the solid support of claim 9, the module comprising a startslot, an end slot and a series of wells disposed between the start slotand the end slot, the module characterised in that the end slot includesa cut-out portion into which the flexible outwardly extending arms ofthe solid support snap-fit.
 19. The module of claim 14, wherein the endslot is provided with a window through which results obtained with thesolid support may be read either manually or through automated means.20. A machine for use with a dipstick according to claim 3 and a moduleas defined above, wherein the machine is characterised by a reader meansfor reading the regions of the solid support, said reader being arrangedto move horizontally only in the machine, with the solid supports beingraised and lowered on a generally vertical axis to present the variousregions of the solid support to the reader means.
 21. The machine ofclaim 20, wherein the reader means comprises a light or reflectancedetector and one or more light sources.
 22. A machine for use with thesolid support of any one of claim 1 and the module as defined above,wherein the machine is characterised by the solid support beingautomatically and sequentially moved to and lowered into and raised fromwells in the module in sequence with the solid support remaining in eachwell for a predetermined period of lime.
 23. The machine of claim 22,wherein the machine includes a head defining a gripper means forgrasping a top portion of the solid support.
 24. The machine of claim23, wherein the movement of the head and hence the solid support iscontrolled to suit particular assays.
 25. A machine according to claim24, wherein movement of the head is controlled by programming themachine through the use of a smartcard and smartcard reader.